Purification methods for monoclonal antibodies generally comprise the following four fundamental steps: (1) collection—separation of host cells from fermentation culture products; (2) capture—separation of antibodies from most components of purified collected materials; (3) fine purification—removal of remaining host cell contaminants and aggregates; and (4) formulation—placing antibodies in suitable carriers to ensure maximum stability and storage period of the antibodies. However, these steps do not always produce antibody compositions having sufficient purity for use in pharmaceutical circumstances. Thus, it is particularly important to have a method of producing and purifying a desired antibody having suitable purity for pharmaceutical use.
Protein-A chromatography makes it possible to substantially completely purify antibodies, particularly IgG, from cell culture supernatants by a single step, and thus is widely used in industrial antibody production. However, a protein-A chromatography column has a problem in that a ligand can leak somewhat from the column due to the repeated use of the column. This protein A or protein A fragment has affinity for IgG to form a complex with the antibody so as to contaminate the antibody and is also difficult to remove from a purified antibody. Particularly, because protein A is a bacterial protein, it can induce an undesired immune response, and thus should be removed from a purified antibody. Accordingly, processes of purifying antibodies using protein A chromatography have a problem in that it is required to monitor and remove the remaining protein A in each process.
However, large foreign pharmaceutical companies mostly use protein A chromatography for antibody purification, because protein A chromatography can achieve the high-purity purification of antibodies, even though it is expensive. However, due to the above-described problem, it is needed to develop a process capable of substituting for protein A chromatography. If a highly economic antibody purification process capable of substituting for protein A chromatography were developed, it could be very advantageous in cost terms because it could reduce the process cost by up to 50-70% compared to existing processes, and it could also prevent protein A contamination.
However, in the development of an antibody purification process that does not use a protein A chromatography column, it is important to develop a process that can show impurity removal efficiency comparable to that of a process that uses a protein A chromatography column. Particularly, antibody cultures produced using animal cells, particularly CHO cells, contain, in addition to a desired antibody, large amounts of impurities, such as host cell protein (HCP) and host cell DNA (HCD), derived from the CHO cells, and also contain cell growth factors. Thus, if an antibody is to be purified without using a protein A chromatography column, it is particularly important to determine the amount of impurities that can be removed in an initial stage. However, in order to develop an antibody purification process that can result in a low HCP level without using protein A chromatography, it is required to determine the kind and order of suitable chromatography processes and to develop procedures for optimization of processes, but it is difficult to achieve this determination and development.